Oxygen removal

ABSTRACT

An oxygen scavenging composition for use with foods, cosmetics, and pharmaceuticals comprising a combination of a metal that can exist in two redox states at ambient conditions, and a radical oxygen scavenger. The oxygen scavenging system is also effective in inhibiting the growth of yeasts, molds, and most aerobic bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 07/279,604,filed Dec. 5, 1988, now abandoned, which was a continuation ofapplication Ser. No. 07/100,97l, filed Sep. 25, 1987, now abandoned.

FIELD OF THE INVENTION

The present invention relates to a process for removing oxygen fromenclosed spaces.

BACKGROUND OF THE INVENTION

A change in the mechanism of food distribution has brought about anincreasing demand for packaged foods. While packaged food is susceptibleto deterioration in a variety of ways, depending upon conditions duringdistribution and storage, one of the most serious problems arises fromspoilage due to oxygen. A wide variety of packaged foods are caused todeteriorate by oxidation since they are readily oxidizable. Oxidationcauses changes in color and flavors, reduction in nutritional value, andother disagreeable conditions which give rise to complaints fromconsumers.

The changes in quality of packaged foods can also be caused bymicroorganisms. In order to prevent changes due to microorganisms oroxidation, it is customary to rely on food additives, such asantimicrobial and antimycotic agents and antioxidants.

Current methods for removing oxygen from packages are fraught withshortcomings:

1. Flushing with inert gases results in incomplete oxygen removal,particularly in the interior of porous foods. Furthermore, inert gasflushing provides no protection against subsequent oxygen influx. Inorder to flush with inert gases, the package interiors are evacuated andnitrogen or carbon dioxide gas is sealed in the evacuated package.Another problem with this method of preventing oxidative deteriorationof foods is that an exact selection of combinations of articles to bepackaged, and packaging material are required to achieve the intendedresults. Selection of favorable conditions for the methods demands atremendous amount of time and data, and use of a high-efficiencypackaging machine and a highly gas-impervious packaging material.

2. Vacuum packaging can only be used for certain foodstuffs, becausesome foodstuffs are likely to be deformed in the negatively pressurizedinterior of the package.

3. Coupled enzyme systems, such as glucose oxidase/peroxidase, undergorapid inactivation and are very sensitive to changes in pH, wateractivity, solvent system, salt content, temperature, and various otherfactors. Additionally , these systems require the addition of water fromthe outside for their action, and therefore cannot be effectively usedfor low-water content foodstuffs, although they may work reasonably wellwith foodstuffs containing a great amount of water. These systems thushave limited practical utility.

4. Packages containing elemental iron remove oxygen by virtue ofrusting, but at a very slow rate. Thus, this is impractical. This methodis virtually ineffective below 0° C., and therefore provides noprotection against common oxidative freezer damage.

5. The removal of oxygen by hydrogen gas is expensive and burdensome.The material to be protected is packaged in a material of a laminatedstructure of a polyester/metal foil/Surlyn/palladium/Surlyn (trademarkof ionomer by DuPont Company) by gas substitution with a mixture ofhydrogen and nitrogen gases whereby oxygen remaining in the package isreacted with the hydrogen gas under the catalytic action of palladium inthe laminate to permit elimination of oxygen.

Addition of an antioxidant, antiseptic, or any other like additives tofoodstuffs, which has been extensively adopted for the purposes ofpreservation, has the disadvantage that a technically sufficient amountof additives is prohibited by various statutes and regulationsconcerning additives for foodstuffs, pharmaceuticals, or cosmetics inlight of their adverse effects on the human body. In addition, knownhazardous materials cannot be used for foods, pharmaceuticals, andcosmetics.

The sensitivity of ascorbic acid to copper-catalyzed oxidation wasrecognized immediately after its discovery by Szent-Gyorgyi in 1928, andits structural determination and chemical synthesis in 1933. During thepast 50+ years, a multitude of studies were conducted to investigate thekinetics and thermodynamics of oxidation of ascorbic acid by transitionmetals such as copper and iron, and their various chelates.

Historically, trace amounts of copper (less than 0.1 ppm) have beenknown to catalyze oxygen radical formation and lipid peroxidation,leading to rapid food spoilage, especially of food susceptible tooxidative damage. Therefore, painstaking efforts are being made inseveral food areas, such as the dairy industry, to eliminate allexposure of food to copper-containing equipment. Higher levels of copperare expected to aggravate this deteriorative effect of copper. Based onthis current knowledge, the system of the present invention is notobvious and is contrary to conventional wisdom, since 5-7 ppm copper inthe presence of a reducing agent, such as ascorbic acid, completelypreserves oxygen-sensitive foods.

Nakamura et al., in U.S. Pat. No. 4,384,972, disclose an agent formaintaining the freshness of a packaged foodstuff comprising a salt ofmanganese (II), iron (II), cobalt (II), or nickel (II), an alkalicompound, and a sulfite or deliquescent substance. Ascorbic acid or asalt thereof may optionally be included.

Siegel, in U.S. Pat. No. 3,320,046, discloses a formulation forconditioning cut flowers comprising an inorganic compound selected fromthe group consisting of water soluble inorganic salts or chelates whichcontain the one of the following metal ions: copper (II), zinc (II),manganese (II), cobalt (II), or nickel (II). The second component of thecomposition is ascorbic or isoascorbic acid, and the third component ofthe composition is an antioxidant such as a vinyl ether, an alkylphenol, a phenolic ether, or the like.

Pottier, in U.S. Pat. No. 3,294,825, discloses an antioxidantcomposition for protecting lipids against oxidation comprising acombination of ascorbic acid and citric acid.

Stone, in U.S. Pat. No. 2,892,718, discloses a composition for treatingmalt beverages comprising sodium hydrosulfite and a salt of ascorbicacid.

Japanese patent 55-118344 discloses a method for preventingdiscoloration by immersing the vegetables in an aqueous solutioncontaining an acid, a chelating agent such as sodium metaphosphate, anda harshness-removing agent such as burnt alum.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the above-mentioneddeficiencies in the prior art.

It is another object of the present invention to provide a system forreducing oxidative damage to packaged materials such as foods,pharmaceuticals, and cosmetics in a safe manner that does not affect thetaste.

It is yet another object of the present invention to provide a systemfor preserving materials such as foodstuffs, pharmaceuticals, andcosmetics from microbial contamination.

The oxygen-removing composition of the present invention, also referredto as Oxysorb, comprises a combination of a reducing agent, from here oncalled an oxygen scavenger, and a small amount of a transition metalwhich can exist in two valence states, such as copper. Theoxygen-removing system may be dissolved in or otherwise mixed thoroughlywith the material to be protected from oxidation without producing anyharmful effects, or it may be added in a small oxygen-permeable pouchcontaining the system and a suitable solvent.

The oxygen-absorbing system of the present invention protects somefoods, ingestibles, and topical compositions against oxygen-mediatedgeneration of off-flavors and odors, discoloration, enzymatic browning,loss of texture, syneresis, rheological changes, and growth of aerobicbacteria, yeasts, and molds. The system is effective at refrigerator,freezer, and room temperatures.

Oxygen removal occurs with any easily oxidizable reducing agent (oxygenscavenger) such as ascorbic acid. Hereinafter, the reducing agent willbe exemplified by ascorbic acid. Most of these reactions are very slowand must be accelerated by light (as in the case of FMN or flavinmononucleotide) or by a transition metal capable of existing in 2valence states. For exemplary purposes, the transition metal used iscopper. The term copper denotes a species of copper which is soluble inthe system in which it is used, preferably salts of Cu⁺ or Cu²⁺.Therefore, copper functions as a catalyst, cycling between Cu⁺ and Cu²⁺,while never being consumed. The reducing agent, however, is used up andtherefore determines the total oxygen depletion capacity.

Although many transition metals catalyze this reaction, copper is thepreferred substance in foods, since it effects oxygen removal withoutthe concomitant generation of highly reactive oxygen radicals. Manyother metals in the presence of ascorbic acid promote lipid peroxidationand food deterioration. However, they may still be added to foods whencontained within a pouch (see below). In addition to completelypreserving the color, flavor and texture of foods, Cu²⁺ -ascorbate alsoinhibits the growth of aerobic microorganisms presumably by forminghypochlorous acid (HOCl). Other metals tested fail to produce thiscompound and exhibit no measurable bactericidal effects.

The removal of oxygen by the system of the present invention can bedescribed as follows, with ascorbic acid (AA) given as the example ofthe oxygen scavenger:

Ascorbic acid, vitamin C, reduces Cu²⁺ to Cu⁺ to form dehydroascorbicacid (equation I). Cuprous ions (Cu⁺) form a complex with oxygen, and anelectron transfer occurs to give cupric ions (Cu²⁺) and superoxide anionradicals (equation II). In the presence of copper, the latter radicalsrapidly disproportionate to oxygen and hydrogen peroxide (equation III).The complex of copper-ascorbate rapidly reduces the hydrogen peroxide towater (equation IV), without the concomitant generation of hydroxylradical (equation VI), a highly reactive oxidant. The net reaction(equation V) effects complete oxygen removal from an aqueous solutionwithin 3 minutes as shown in FIG. 1. ##STR1## where DHAA = droascorbicacid.

This reaction mechanism has been deduced from the following lines ofexperimental evidence:

The generation of superoxide anion radical (O₂ ⁻) by equation II hasbeen demonstrated by the substantial acceleration of ascorbic acidoxidation by superoxide dismutase (FIG. 2), an enzyme that catalyzesequation III. By disproportionating O₂ ⁻ to H₂ O₂ and O₂ this enzymeincreases the steady-state concentration of H₂ O₂, which then oxidizesascorbic acid. Similarly, inhibition by catalase (FIG. 2), an enzymethat catalyzes equation VII, confirms the formation of H₂ O₂ during theremoval of oxygen by the system of the present invention. If there wereno hydrogen peroxide generated during reaction V, this enzyme would haveno effect.

In past studies, Cu²⁺ has been found to exhibit some superoxidedismutase activity, i.e., it catalyzes the conversion of superoxideanion radical to hydrogen peroxide, whereas Fe³⁺ lacks this effect. Thisdifference partly explains why copper is the preferred metal for Oxysorbused in solution in edible systems, because the superoxide anion radicalis another potentially dangerous activated oxygen species.

Hydrogen peroxide is very stable, even in the presence of Cu²⁺ (resultnot shown). Cu²⁺ -ascorbate, however, degrades hydrogen peroxide veryrapidly, as shown by the large stimulatory effect of H₂ O₂ on theoxidation of Cu²⁺ -ascorbate (FIG. 3). Thus, Cu²⁺ -ascorbate rapidlyreduces hydrogen peroxide to water (equation IV) without the concomitantproduction of hydroxyl radical (equation VI) or oxygen (equation VII).No generation of O₂ ⁻ or .OH could be detected under a variety ofconditions.

    H.sub.2 O.sub.2 +Cu.sup.+ →.OH+OH.sup.- +Cu.sup.2+  (VI)

    H.sub.2 O.sub.2 +Cu.sup.2+.→H.sub.2 O+1/2O.sub.2+Cu.sup.2+(VII)

The system of the present invention has been found to rapidly removehydrogen peroxide, a potentially dangerous oxidant generated in theabove reactions. Fe³⁺ -ascorbate removes hydrogen peroxide much moreslowly than the system of the present invention (FIG. 4). In addition toreducing H₂ O₂ to water by equation IV, Fe³⁺ -ascorbate also produces asmall amount of hydroxyl radical by the Fenton reaction (equation VIII)as shown in FIG. 5.

    H.sub.2 O.sub.2 +Fe.sup.2+→OH.sup.- +.OH+Fe.sup.3+  (VIII)

To summarize, the removal of oxygen by the system of the presentinvention produces an intermediate which is rapidly reduced by copperascorbate.

The presence of copper in the Oxysorb system produces no hydroxylradical, a very potent and indiscriminate oxidant, whereas iron doesproduce this radical, cf. equation VIII.

Cu²⁺ catalyzes the reduction of the superoxide anion radical to hydrogenperoxide, whereas iron does not.

The advantage of the Oxysorb system is that the oxygen is rapidlyremoved without the production of any radicals.

The copper in the Oxysorb system functions as a catalyst in reducingoxygen to O₂ ⁻ (equation II), and then catalyzing the reduction of O₂ ⁻to hydrogen peroxide (equation III). Copper-ascorbate catalyzes thereduction of hydrogen peroxide to water (equation IV). The amount ofascorbic acid determines the total oxygen removing capacity. Ascorbatereduces Cu²⁺ to Cu⁺ (equation I), reduces hydrogen peroxide to water(equation IV), and scavenges and inactivates any potential radicals inthe food system.

Conventional wisdom dictates keeping even trace amounts of copper awayfrom any oxygen-sensitive material, since copper is an excellentcatalyst for free radical generation, lipid peroxidation, and subsequentputrefaction of foods. However, the system of the present invention doesnot appear to adhere to conventional wisdom for several reasons:

1. Oxygen is absent during most of the shelf-life of the food, since itis rapidly depleted by the system of the present invention immediatelyafter the components are mixed. Oxygen amounts exceeding the capacity ofthe system should be avoided by reducing the headspace or partiallyflushing with an inert gas. Otherwise O₂ and Cu²⁺ (in the absence of anyremaining ascorbic acid) will cause food spoilage.

2. The relatively high concentration of copper used in the invention (˜5ppm) catalyzes the rapid reduction of O₂ ⁻ to H₂ O₂.

3. The relatively high concentration of copper used in the invention(around 5 ppm) catalyzes the rapid breakdown of hydrogen peroxide toform water, which is inert.

In addition to inhibiting oxidative rancidity and other oxygen-mediateddeteriorative reactions, the system of the present invention alsoappears to reduce spoilage by suppressing growth of aerobicmicroorganisms, both through oxygen deprivation and direct bactericidalaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the oxygen utilization by the copper-ascorbate oxygenscavenger system. The oxygen tension was measured in 1 mM sodiumascorbate and 50 mM Tris, pH 7.4 at 25° C. using a Clark oxygenelectrode.

FIG. 2 shows the effects of 1000 U/ml catalase and 25 U/ml superoxidedismutase (SOD) on the oxidation of 50 micromolar ascorbic acid todehydroascorbic acid in the presence of 1 micromolar Cu²⁺. The rate ofoxidation at 25° C. in 50 mM Tris, pH 7.4, was monitoredspectrophotometrically.

FIG. 3 shows the effect of 100 micromolar H₂ O₂ on the oxidation of 50micromolar ascorbic acid to dehydroascorbic acid in the presence of 1micromolar Cu²⁺. The rate of oxidation at 25° C. in 50 mM Tris, pH 7.4,was monitored spectrophotometrically.

FIG. 4 shows the effects of 1 micromolar Cu²⁺ and 10 micromolar Fe³⁺ onthe oxidation of 50 micromolar ascorbic acid. Both reactions werecarried out at 25° C. in 50 mM Tris, pH 7.4.

FIG. 5 shows the effect of Fe³⁺ on the generation of hydroxyl radical(.OH) at 25° C. in 50 mM Tris, pH 7.4, 2 mM H₂ O₂, 2 mM ascorbic acidand 50 mM DMSO. Hydroxyl radical was determined by quantitatingformaldehyde formed from the reaction between .OH and DMSO.

FIG. 6 shows the effect of water activity (a_(w)) on oxygen utilizationby the system at 25° C. Solutions of variable water activity containing40 ppm copper gluconate and 0.2% ascorbic acid in 50 mM Tris, pH 7.4,were stored in oxygen-impermeable plastic tubs for 28 days. Headspaceoxygen was withdrawn with a hypodermic syringe and analyzed using anoxygen analyzer. Variable water activities were achieved by saturatingthe solutions with the following salts: LiCl (0.1), MgCl₂ (0.37), NaBr(0.59), NaCl (0.75), KCl (0.83), K₂ SO₄ (0.93), no salt (1.00).

FIG. 7 shows the effect of propylene glycol on copper-mediated oxygenutilization. Initial rates were measured in 50 micromolar coppersulfate, 1 mM sodium ascorbate, and 50 mM Tris, pH 7.4, at 25° C.

FIG. 8 shows the effect of temperature on the rate of oxygen diffusioninto an Oxysorb pouch. A polyethylene pouch (48 cm²) containing 22 ml ofOxysorb was sealed inside a 6 oz. oxygen-impermeable tub. Headspace O₂was determined after various time intervals; each time point representsa different sample.

FIG. 9 shows the effect of the Oxysorb addition method (directdissolution vs. pouch addition) on the rate of oxygen removal fromsolution and headspace.

FIG. 10 shows the oxygen gradient between solution and headspace for thepouch method versus direct addition of Oxysorb.

FIG. 11 shows the effect of ascorbic acid concentration on the rates ofCO₂ generation and O₂ utilization at pH 7.4.

FIG. 12 shows the effect of ascorbic acid concentration on the rates ofCO₂ generation and O₂ utilization at pH 3.0.

FIG. 13 shows the effect of copper concentration on oxygen utilizationin 1 mM sodium ascorbate and 50 mM Tris, pH 7.4, at 25° C. Each pointrepresents the initial slope of oxygen curves shown in FIG. 1 (initialrates). The solid line shows the best fit calculated from a linearregression analysis.

FIG. 14 shows the effect of oxygen tension on oxygen utilization atvarious concentrations of copper. The experiment were carried out at 25°C. in 1 mM sodium ascorbate, 50 mM Tris, pH 7.4, and increasingconcentrations of copper sulfate.

FIG. 15 shows the effect of ascorbate concentration on copper-mediatedoxygen utilization. Each experiment was carried out at 25° C. in 50micromolar copper sulfate, 50 mM Tris, pH 7.4, and increasing amounts ofsodium ascorbate.

FIG. 16 shows the effect of pH on copper-mediated oxygen utilization in100 micromolar copper sulfate and 2 mM sodium ascorbate at 25° C. Thefollowing buffers were used to achieve the desired pH values: aceticacid, pH 4.0; acetic acid, pH 5.0; imidazole, pH 6.0; imidazole, pH 7.0;Tris, pH 7.4; Tris, pH 8.0; glycine, pH 10.0.

FIG. 17 shows the Arrhenius plot of copper-mediated oxygen utilization.The initial rates were determined in 50 micromolar copper sulfate, 1 mMsodium ascorbate, and 50 mM Tris, pH 7.4 at 10° C., 15° C., 20° C., 25°C., 30° C., 35° C., and 40° C.

FIG. 18 shows the effect of chelating agents on oxygen removal byOxysorb. The rate of O₂ consumption at 25° C. in 10 mM sodium ascorbate,100 micromolar copper gluconate, 50 mM Tris, pH 7.4, and 1 mM chelatingagent was monitored using a Clark electrode.

FIG. 19 shows the effect of different transition metals on oxygenremoval. The rate of O₂ consumption at 25° C. in 10 mM sodium ascorbate,50 mM Tris, pH 7.4, and 100 micromolar transition metal was monitoredusing a Clark electrode.

FIG. 20 shows the effect of different reducing agents on copper-mediatedoxygen removal. The rate of O₂ consumption at 25° C. in 100 micromolarcopper gluconate, 50 mM Tris, pH 7.4, and 1 mM reducing agent wasmonitored using a Clark electrode.

FIG. 21 shows the effect of protein on O₂ removal by Oxysorb. Increasingamounts of ovalbumin in the absence and presence of glycine were addedto the Oxysorb system and the rate of O₂ consumption at 25° C. wasmonitored using a Clark electrode.

FIG. 22 shows the effect of Oxysorb on the oxidation of emulsions.Samples were stored in oxygen impermeable plastic jars at roomtemperature and analyzed intermittently for malondialdehyde (MDA).

FIG. 23 shows the effect of Oxysorb on the oxidation of oils. Sampleswere stored in oxygen impermeable plastic jars at room temperature andanalyzed intermittently for malondialdehyde (MDA).

FIG. 24 shows the effect of Oxysorb on the oxidation of emulsions.Samples were stored in oxygen impermeable plastic jars at roomtemperature and analyzed intermittently for lipid peroxidation.

FIG. 25 shows the effect of Oxysorb on the oxidation of oils. Sampleswere stored in oxygen impermeable plastic jars at room temperature andanalyzed intermittently for lipid peroxidation.

FIG. 26 shows the effect of Oxysorb on polyphenol oxidase activity. Theoxidation of 0.3 mg/ml DOPA by 0.02 mg/ml polyphenol oxidase at 30° C.in the absence and presence of 40 ppm copper gluconate / 0.2% ascorbicaid was monitored spectrophotometrically.

FIG. 27 shows the effect of 0.004% copper gluconate and 0.2% sodiumascorbate on the growth of E. coli at 25° C. under aerobic and anaerobicconditions.

FIG. 28 shows the effect of the oxygen scavenging system (0.004% coppergluconate and 0.2% sodium ascorbate) on yeast growth at 25° C.

FIG. 29 shows the effect of 0.004% copper gluconate and 0.2% sodiumascorbate on oxygen depletion in the headspace of Mexican salsas. Thesolid lines represent theoretical oxygen utilization rates calculatedform first order kinetic parameters. All products were stored at 5° C.

FIG. 30 shows the effect of Oxysorb on O₂ removal from the headspace ofpizza sauce stored at -12° C. Shredded cheese was dispersed in the sauceto increase its susceptibility to oxidative discoloration.

FIG. 31 shows the dependence of browning of potato water on Oxysorb(0.1% ascorbic acid and 0.004% copper gluconate) at 5° C.

DETAILED DESCRIPTION OF THE INVENTION

The Oxysorb system of the present invention may be added directly to afood product which is to be preserved, or may be added to a food productin an oxygen-permeable pouch.

The total capacity for oxygen removal is determined by the amount ofascorbic acid. The complete 2-step reduction of 1 mole of oxygen towater requires 2 moles of ascorbic acid. This stoichiometry may betranslated into the following equalities:

    ______________________________________                                        1 liter of air at 20° C.                                                             =      18.75    mmoles ascorbate                                1 liter of water at 20° C.                                                           =      0.50     mmoles ascorbate                                              =      0.099    g sodium ascorbate                                            =      0.01%    sodium ascorbate in                                                           water                                           ______________________________________                                    

For the above calculations we assumed an oxygen solubility of 0.25 mM inwater at 20° C.; this was the experimentally determined oxygensolubility in 50 mM Tris, pH 7.4 at 20° C. The exact requirement forascorbic acid in a food system will depend on the oxygen solubility inthat particular solution or solid.

The total oxygen depletion capacity of the oxygen absorbing system isdirectly proportional to the amount of reducing agent in the system. Theminimum concentration of reducing agent should be calculated for eachapplication. An excess of reducing agent such as ascorbic acid merelyprovides longer protection, and has no detrimental effect on the foodsystem.

The total amount of ascorbic acid or other reducing agent required toprovide full protection throughout the shelf life of a packaged productmay be substantially increased for the following five reasons:

1. If it is necessary to heat process the substrate during itsmanufacture, after the copper and the oxygen scavenger have already beenadded, significant amounts of the oxygen scavenger may become degraded.

2. A slow influx of oxygen into the system over a period of time willrequire more oxygen scavenger for full protection. For example, in thecase of oxygen-permeable packaging materials, oxygen will slowly beadded to the system from outside of the package.

3. The oxygen scavenger in the system not only reduces the copper fromone oxidative state to another, but it also scavenges any deleteriousoxygen radicals. Since both reactions oxidize ascorbic acid, a slightexcess of ascorbic acid is required to provide sufficient radicalscavenging antioxidant activity.

4. As ascorbic acid approaches its depletion, the rate of reduction ofthe metal becomes greatly depressed, since the concentration of ascorbicacid becomes rate-limiting. An excess of ascorbic acid assures rapidoxygen removal even at low oxygen concentrations.

5. Strong oxidizing agents added to certain foods may oxidize a portionof the oxygen scavenger, such as azodicarbonamide or bromate added toflour doughs.

Exposure of the Oxysorb system to oxygen and heat processing should beminimized in order to avoid extensive oxidation of the oxygen scavenger.

With the Oxysorb system of the present invention, oxygen is removed soas to prevent deleterious oxidation. This can be determined by standardtesting methods, e.g., organoleptically in foods.

The system of the present invention can be introduced into the productto be preserved in a variety of ways. Of course, if the product to bepreserved is a liquid, the system can be added directly to the liquid.In the case of a solid product, the system can be contained inside apouch contained in the package of the product. The system may also beincorporated into the lid of a can or a jar, or incorporated into thewrapping material for the product. Alternatively, the drymetal/ascorbate powder can be enrobed in an oxygen-and water-permeablematerial. Another method is incorporation of the metal into thepackaging material and addition of the oxygen scavenger to the product.This combination assures maximum stability of the system withoutcompromising the speed of dissolved oxygen removal.

A particularly convenient method for incorporating the system of thepresent invention into a packaged product is by use of a pouch withinthe package. The metal/scavenger system is dissolved in a small volumeof water inside a pouch consisting of a water-impermeable film with ahigh oxygen diffusivity. The pouch can then be added to the package tocause oxygen depletion.

An alternate version of the pouch contains compartments which areseparated by a weak wall. One of the compartments contains the oxygenscavenger, and the other contains the metal salt, water, and propyleneglycol or other carrier. Upon breaking the wall between the twocompartments, the reactants mix and begin removal of oxygen. Thiscompartmentalized design has the advantage of rendering the pouch itselfshelf-stable in a high oxygen environment for years, until it becomesmechanically activated by breaking the seal by light compression. Theonly disadvantage of this configuration is the relatively slowdissolution of the reactants.

When the oxygen absorbing system of the present invention is used in atwo-compartment pouch, the metal salt may be dissolved in water or anaqueous medium containing a cosolvent such as propylene glycol,glycerol, or ethanol; salts, such as sodium chloride or other flavoringingredients; pH buffers; chelating agents; glucose; gums; and/orpreservatives.

The advantages of use of the pouch over direct dissolution of the systemin the product to be treated are as follows:

1. The redox reactions between the metal, oxygen scavenger, and oxygentake place inside the pouch, so that no radicals are released into thefood (in the case of Fe³⁺), and the scavenger is not in direct contactwith reducible dyes, such as anthocyanins and azo dyes. These compoundsundergo a color change upon reduction by ascorbic acid.

2. The pouch can be added after the product has been heat-treated.

3. Use of a pouch is particularly useful for products having low wateractivity. The system removes O₂ at a slower rate when it is addeddirectly to a food product having a water activity less than 0.8, (FIG.6).

4. The pouch can be used in packages where the product is subjected tofreezing conditions, i.e., below 0° C. The Oxysorb system was found tobe completely inactive in ice, so it may be ineffective in frozen foodsthat contain little bound water. In this case, wherein lipidperoxidation is often the predominant mode of product failure, themetal/ascorbate system is dissolved in 40% propylene glycol and added tothe food in a pouch. The rate of oxygen removal was not affectedsignificantly by the addition of this freezing point depressant (FIG.7). Propylene glycol is used because of its low viscosity, lack oftoxicity, and low cost, although other freezing point depressants canalso be used. A corrugated surface may be provided for the package toincrease the surface area thereof, accelerating diffusion of oxygen intothe pouch.

A study was conducted to determine the rate of oxygen removal by pouchescontaining the system of the present invention as a function oftemperature, and to compare this pouch method to the direct dissolutionmethod.

A solution was prepared containing 13.7% ascorbic acid, 40 ppm coppergluconate, 50 mM Tris buffer at pH 7.4, and 25% propylene glycol toprevent freezing. The pouches were an average size of about 6×4 cm (48cm²) and contained about 22 ml of the solution. The polyethylene pouchfilm was a high water barrier and a low oxygen barrier. The pouches werefilled and sealed to contain no headspace. Each pouch was then sealedinside a 6 oz. polypropylene tub with an aluminum foil lid. The sampleswere divided equally and stored at 100° F. (38° C.), 70° F. (21° C.),40° F. (5° C.), or -10° F. (-24° C.). Headspace oxygen evaluations weredone intermittently, depending upon the storage temperature and thepredicted rates.

FIG. 8 illustrates the rates of oxygen depletion using the pouches atdifferent temperatures. The rate of the reaction is faster the higherthe temperature. The removal rate depends on temperature, pouch area,initial oxygen concentration, and it is independent of the headspace(i.e., a given pouch removes 10 ml at the same rate as 100 ml).

Although the pouches were found to work in the tested range oftemperatures, the rates of oxygen removed from the headspace are roughlyhalf those of direct addition, as shown in FIG. 9. Furthermore,dissolved oxygen is removed within one minute by direct addition, whileit is removed at the same rate as headspace oxygen (approximately 10days) in the pouch method, cf. FIG. 9.

This large difference arises from the opposite driving forces in the twoexperiments. In the direct addition method, the dissolved oxygen tensionis zero after one minute. This sets up a large, steep oxygen gradientacross the air-liquid interface which drives oxygen from the headspaceinto the solution, cf. FIG. 10. In the pouch method, however, the oxygentension in solution and headspace remain virtually identical throughoutshelf-life. Slow oxygen removal from the headspace by the pouch createsan infinitesimal gradient, which forces reequilibration betweenheadspace and solution by slow movement of oxygen from solution toheadspace, as shown in FIG. 10.

The addition of the system of the present invention also provides afoolproof and rapid method for detecting leaks and misformulatedbatches. Samples that contain both copper and ascorbic acid and thathave been sealed properly develop a strong vacuum after 24 hours. Thealuminum foil packaging is very tight and concave.

It has been noted that when the system of the present invention is addedto a food system in a pouch, the package changes shape with differentstages of the reaction. Within one day, a vacuum is created as theoxygen is depleted. As the reaction proceeds, the vacuum decreases.After about 20 to 28 days, a ballooning effect is evident. Apparently,the reaction produces carbon dioxide in excess of the depletion ofoxygen. One of the indicators of this reaction is a color change of thesystem from a clear liquid initially to a dark brown.

Trials were conducted to determine the concentration of carbon dioxideproduced and to ascertain if pH and ascorbic acid concentrationinfluence the amount of carbon dioxide production.

Ten ml of a solution of 40 ppm copper gluconate and variableconcentrations of ascorbic acid were sealed in a 6 oz. empty,oxygen-impermeable tub and stored in the dark at ambient temperature.The tubs were then analyzed for oxygen, carbon dioxide, and nitrogen ona gas chromatograph. The variables were:

pH 7.4 with 15% ascorbic acid, 10% ascorbic acid, 5% ascorbic acid, 0.2%ascorbic acid;

pH 3.0 with 15% ascorbic acid, 10% ascorbic acid, 5% ascorbic acid, and0.2% ascorbic acid.

FIGS. 11 and 12 show that the concentration of ascorbic acid had nosignificant effect on the rate of oxygen utilization, the rate ofdecarboxylation, and the total amount of carbon dioxide produced. Thisindicates that diffusion of oxygen into the liquid is the rate-limitingstep, and the quantity of carbon dioxide formed depends strictly on theamount of oxygen available, i.e., each molecule of ascorbic acid thathas been oxidized by oxygen to dehydroascorbic acid undergoes a fast andsubsequently a slow decarboxylation to form a product of unknownstructure. The second decarboxylation is responsible for the largepositive pressure inside food packages containing a pouch. Ballooningappears as soon as the combined amount of carbon dioxide and remainingoxygen exceed 21% of the headspace.

Development of positive pressure in food packages containing a pouchaccording to the present invention occurs primarily with foods of verylow carbon dioxide solubility, such as solids. Flushing with nitrogen ordecreasing the headspace to reduce the initial oxygen level are twoapproaches to minimizing this problem. However, in some cases, highlevels of carbon dioxide may be beneficial, as they are toxic to manymicroorganisms.

A concern was raised over the amount of water generated by the system ofthe present invention, particularly in dry foods. However, it was foundthat 0.2% ascorbic acid in any food was calculated to produce only0.018% moisture, while it depletes a substantial quantity of oxygen. Onegram of a food containing 0.2% ascorbic acid has the capacity to removeall of the oxygen from 0.61 ml air. Therefore, the system of the presentinvention has no noticeable effect on the moisture of even very dryfood, such as corn chips.

The blue color of the copper salts does not affect the visible color ofthe food at the very low concentrations used in the present invention.Both ascorbic acid and copper contribute no odor or flavor to the food.The pH remains unchanged in most food systems because of the food'sbuffering capacity.

With regard to current FDA regulations, copper may be added to foods andpharmaceuticals for oral ingestion in the form of copper gluconate as adietary supplement not exceeding 50 ppm (equivalent to 7.0 ppm copper),or as a processing aid not exceeding good manufacturing practices.Ascorbic acid can be labelled as vitamin C or as ascorbic acid,depending upon the type of food or pharmaceutical product involved.

At a pH of 7.4, 90% of dissolved oxygen is removed by 100 micromoles ofcopper sulfate (6.4 ppm copper) and 2 mM sodium ascorbate (0.040%)within 1.8 minutes, as shown in FIG. 1. No oxygen utilization isobserved in the presence of copper alone or ascorbate alone. The initialrate of oxygen removal in the presence of copper and ascorbate wascalculated to be 133 mmHg/min. As discussed previously, the mechanismfor oxygen removal by copper and ascorbate consists of a reduction ofCu²⁺ to Cu⁺ by ascorbate, followed by a reduction of oxygen by Cu⁺ toregenerate Cu²⁺.

The rate of this reaction is linearly dependent upon the copperconcentration, as shown in FIG. 13. Even 2 micromolar Cu²⁺ catalyzesmeasurable oxygen consumption. The concentration of the copper in thesystem of the present invention can vary over a wide range, from about 1ppm to about 70 ppm in the system being protected, the upper limit alsobeing dependent upon FDA limitations in upper centrations on food.However, for optimum performance, a minimum of at least 3 ppm isrecommended. At low pH, i.e., at pH less than 6, the concentration ofthe copper is preferably at least 6 ppm.

The rate is also directly proportional to the oxygen tension over a widerange of copper concentrations, as shown in FIG. 14.

Ascorbic acid affects the rate of oxygen depletion primarily at lowconcentrations, as seen in FIG. 15. At high concentrations, thedependence is partly due to increasing transition metal contamination.

As can be seen from FIG. 16, the rate of oxygen utilization increaseswith pH. The two exceptions at pH 6.0 and 8.0 may arise fromcopper-buffer interactions, since several of the pH buffers form weakchelates with copper.

Oxygen utilization is fairly temperature-dependent. From the Arrheniusplot shown in FIG. 17, a temperature coefficient (Q₁₀) of 2.0 wascalculated, which means that at a freezer temperature of -12° C. therate will be 6% of that at 25° C.

The Oxysorb system of the present invention is also effective in thepresence of chelating agents, such as citric acid, EDTA, phosphatecompounds, L-histidine, glycine, and mixtures thereof. The chelatingagents used for this experiment were glycine, L-histidine, phytic acid,and EDTA. Purified water and Tris were dispensed into the vial, and then1 mM chelating agent and 100 μM copper gluconate were added. Uponstabilizatioh the oxygen electrode, the ascorbic acid was introduced andthe reaction quickly took place. As illustrated in FIG. 18, in the caseof glycine, the initial rate was faster than that of the control, whichcontained no chelating agent. Phytic acid was slightly slower than thecontrol. Histidine caused some inhibition. EDTA almost completelyinhibited the reaction.

Another experiment was designed to substitute a variety of transitionmetals and reducing agents in place of copper and ascorbate. Thetransition metals were ferric chloride, manganese sulfate, cobaltchloride, and chromium chloride. The final concentration of these metalswas 100 micromolar in 10 mM ascorbic acid and 50 mM Tris buffer, pH 7.4.To start the reaction, the metal was added to these solution, and theremoval of dissolved oxygen was monitored using a Clark electrode.

Referring to FIG. 19, it is evident that the copper gluconate control isthe best catalyst for this reaction. However, the other metals dodemonstrate the ability to promote the oxygen removal reaction, althoughthe initial rates are significantly slower.

The reducing agents substituted for ascorbic acid were sodium sulfite,cysteine, and catechol. The order of addition was purified water, Tris,and 1 mM reducing agent. When the electrode stabilized, the coppergluconate was added. FIG. 20 illustrates that the ascorbic acid controlis the most efficient reducing agent compared to the substitutes.Nevertheless, the alternatives were somewhat effective, cysteine beingmore reactive than sulfite and sulfite being more reactive thancatechol.

The system of the present invention was evaluated to determine theeffectiveness of the system in various water activity systems.

Six salt solutions were prepared to provide a range of water activitiesbetween 0.1 and 1.0. The system of the present invention was added, andthe solutions were sealed in oxygen-impermable containers. The headspaceof these containers was analyzed for % oxygen over a period of 28 days.

From the results shown in FIG. 6, the system of the present inventionwas significantly faster at high water activities. In systems with awater activity lower than 0.8, it may be advisable to add the systemencased in a pouch. A benefit of this great dependence on water activityis the stability of the Oxysorb system in a dry system. Thus, copper andthe oxygen scavenger may be incorporated into a dry preblend and storedfor several months before being added to the remaining food components.

Experiments were conducted to determine the effect of protein on thereaction of the Oxysorb system. Using a Clark electrode, the oxygenconsumption was measured in solutions containing 50 mM Tris, pH 7.4,0.2% ascorbic acid, 40 ppm copper gluconate, variable amounts ofovalbumin, with and without 2 mM glycine. The order of addition waswater, Tris, glycine, copper gluconate, ovalbumin, and ascorbic acid.

FIG. 21 shows that ovalbumin binds copper with a high affinity andthereby greatly decreases the rate of oxygen removal. The addition ofglycine, a chelating agent, diminished this protein effect by competingfor copper and making it available for reduction by ascorbic acid.Despite the great inhibitory effect of protein, the system of thepresent invention still effects complete oxygen removal within 25minutes at 25° C. However, it may be desirable to add a chelating agent,such as glycine, to certain types of foods that already have depressedOxysorb activity due to low a_(w) /temperature or high viscosity.

A number of oil-based food systems undergo oxidative rancidity anddevelop off-flavors, so that a system to scavenge oxygen from suchsystems would aid in preserving such foods. For such a system, afat-soluble derivative of the ascorbic acid/copper combination was used.Oxidative damage was measured by peroxide value (PV) and thethiobarbituric acid (TBA) test for malondialdehyde (MDA).

The fat-soluble system of the present invention comprisesascorbyl-6-palmitate in absolute ethanol and copper caprylate inethanol. The final concentrations were 0.47% ascorbyl palmitate and 30.8ppm copper caprylate in the emulsions.

Four different emulsions and oils were used: water-in-oil (w/o),oil-in-water (o/w), liquid shortening, and solid shortening. None of theoils contained antioxidants such as BHA. Xanthan gum (1%) and sodiumstearoyl lactylate (0.5%) were added to the o/w to thicken and emulsify,respectively. Mono-diglycerides were added to the w/o emulsion tostabilize it. The two oils did not contain any emulsifiers.

The emulsions were prepared and dispensed into polypropylene tubs andsmall 60 ml oxygen-impermeable plastic jars with no headspace. Theemulsions were stored at ambient temperature and evaluatedintermittently. The tubs were used for headspace oxygen analysis, andthe jars were evaluated for PV and MDA.

The MDA values of all emulsions and oils containing the Oxysorb systemnever exceeded 0.5, whereas the controls increased to 2.8 mg of MDA/1,as shown in FIGS. 22 and 23. Similarly, the peroxide value of allemulsions and oils was substantially reduced by the addition of Oxysorb,shown in FIGS. 24 and 25. These studies demonstrate thatcopper-ascorbate stabilizes both aqueous and oil systems and protectsthem against oxidative damage and food spoilage.

It has also been found that the system of the present invention stronglyinhibits the activity of polyphenol oxidase (tyrosinase), the enzymeprimarily responsible for the browning of vegetables and fruits.Bruising, cutting, and processing this type of food acceleratesenzymatic browning by releasing both enzyme and substrates and bringingthem into close vicinity. Since oxygen is absolutely essential for thesereactions to occur, the copper-ascorbate system was shown tosubstantially retard the browning of cut potatoes and of guacamole.

In the following experiment, pure mushroom tyrosinase (polyphenoloxidase) and the substrate DL-dihydroxyphenylalanine (DOPA) were used todemonstrate the inhibition of browning by the Oxysorb system of thepresent invention.

Five hundred microliters of deionized water, 200 microliters of 2%ascorbic acid, and 200 microliters of 400 ppm copper gluconate wereadded to 1000 microliters of 0.6 mg/ml DOPA in 200 mM phosphate, pH 6.5.The mixture was allowed to stand for ten minutes to ensure that all ofthe oxygen had been removed. Then 100 microliters of 0.4 mg/ml ofpolyphenol oxidase was added, and the absorption was measuredspectrophotometrically at 475 nm at 30° C. for one hour (read versus thecomplete reaction mixture but without the enzyme). The enzyme iscompletely inhibited by the system of the present invention. The controlreaches an absorbance of 0.36 after 30 minutes, as shown in FIG. 26.

The system of the present invention has also been shown to inhibit thegrowth of a number of microorganisms that are undesirable in food,pharmaceutical, and cosmetic products. To determine the antimicrobialeffects of Oxysorb, 10 ml of broth in 16×100 screw-capped tubes wereinoculated with diluted, overnight cultures to achieve an initialpopulation of 100 to 1000 cells/ml. Nutrient broth was used for E. coliand Pseudomonas fluorescens; fluid thioglycollate medium was used forClostridium sporoqenes. Following inoculation, sodium ascorbate wasadded at a level of 0.2%, then 88 μM metal salt (copper gluconate,cobalt chloride, or ferric chloride). Ascorbate, copper, cobalt, andiron solutions were prepared and filter sterlized just prior to use.Tubes were incubated at 25° C. with caps tightened, unless they wereplaced in anaerobic (Gas Pak) jars. Anaerobically incubated tubes hadloose caps to allow for generation of an anaerobic environment in thetube headspace. Samples from two individual tubes were platedperiodically.

It was found that ascorbate alone had little effect on the growth of E.coli, as shown in Table I. Anaerobic incubation with and withoutascorbate slightly reduced the maximum population, although the growthrate was not affected. Copper alone reduced both the growth rate and themaximum population for E. coli in nutrient broth (Table I). Anaerobicincubation with copper alone further suppressed growth.

The system of the present invention, containing both copper andascorbate, greatly inhibited the growth rate of E. coli in sealed tubes,and completely inhibited the growth of the organism for four days whenthe tubes were incubated in an anaerobic jar, as shown in FIG. 27.

Cobalt and cobalt/ascorbate affected E. coli growth in a manner similarto copper and copper/ascorbate, as shown in Table I. Growth was slowedbut not stopped. Iron and iron/ascorbate, however, had no effect on thegrowth of E. coli.

Pseudonomas fluorescens, an obligate aerobe, responded in a mannersimilar to E. coli (cf. Table II), i.e., growth in the presence of ironor iron/ascorbate did not differ from the control. Cobalt,cobalt/ascorbate, and copper/ascorbate (Oxysorb) slowed the growth ofthe organism. Oxysorb also markedly inhibited yeast growth over a 4-dayperiod (FIG. 28), whereas copper alone showed no toxicity under the sameconditions. Similarly, the system has been found to suppress the growthof Salmonella and Staphylococcus aureus, two other food spoilagepathogens. Conversely, Clostridium sporogenes, an obligate anaerobe, andlactic acid bacteria, were not inhibited by any of the treatments.

                  TABLE I                                                         ______________________________________                                        Growth of E. coli in nutrient broth with and                                  without sodium ascorbate (0.2%) and metals (88 μM sodium).                 Time (days)                                                                             0      0.25    1      2       5                                     ______________________________________                                        Control   180    1800    2.1 × 10.sup.8                                                                 5.5 × 10.sup.8                                                                  2.5 × 10.sup.8                  Ascorbate 350    4600    1.6 × 10.sup.8                                                                 4.6 × 10.sup.8                                                                  3.8 × 10.sup.8                  Fe.sup.3+ 420    6400    2.1 × 10.sup.8                                                                 3.4 × 10.sup.8                                                                  1.5 × 10.sup.8                  Fe.sup.3+ 100Asc.                                                                              11000   6.5 × 10.sup.8                                                                 5.3 × 10.sup.8                                                                  2.7 × 10.sup.8                  Co.sup.2+ 430     690    8.1 × 10.sup.5                                                                 2.2 × 10.sup.6                                                                  4.5 × 10.sup.8                  Co.sup.2+ 430Asc.                                                                               780    8.8 × 10.sup.5                                                                 2.2 × 10.sup.8                                                                  6.6 × 10.sup.8                  Cu.sup.2+ 510    3100    4.7 × 10.sup.7                                                                 4.8 × 10.sup.7                                                                  7.3 × 10.sup.6                  Cu.sup.2+ 700Asc.                                                                               440    1.1 × 10.sup.5                                                                 2.3 × 10.sup.6                                                                  5.5 × 10.sup.7                  (Oxysorb)                                                                     ______________________________________                                    

                  TA8LE II                                                        ______________________________________                                        Growth of Pseudomonas fluorescens in nutrient                                 broth with and without sodium ascorbate (0.2%)                                and metals (88 μM).                                                        Time (days)                                                                             0      0.25    1      4       6                                     ______________________________________                                        Control   150    210     8.1 × 10.sup.7                                                                 1.4 × 10.sup.8                                                                  7.1 × 10.sup.8                  Ascorbate 160    170     4.7 × 10.sup.7                                                                 7.2 × 10.sup.8                                                                  2.7 × 10.sup.8                  Fe.sup.3+ 130    160     6.2 × 10.sup.7                                                                 8.1 × 10.sup.7                                                                  5.1 × 10.sup.7                  Fe.sup.3+ 150Asc.                                                                              280     2.8 × 10.sup.7                                                                 3.3 × 10.sup.7                                                                  1.1 × 10.sup.8                  Co.sup.2+ 150     65     1.9 × 10.sup.6                                                                 5.6 × 10.sup.8                                                                  5.5 × 10.sup.7                  Co.sup.2+ 170Asc.                                                                              170     6.4 × 10.sup.4                                                                 2.0 × 10.sup.8                                                                  1.7 × 10.sup.8                  Cu.sup.2+ 140    180     9.6 × 10.sup.7                                                                 --      1.6 × 10.sup.8                  Cu.sup.2+ 140Asc.                                                                               29     1.1 ×  10.sup.5                                                                7.6 × 10.sup.6                                                                  1.8 × 10.sup.7                  (Oxysorb)                                                                     ______________________________________                                    

The system of the present invention slowed the growth of facultativelyaerobic microorganisms studied by an unidentified mechanism, but had noeffect on aerotolerant lactic acid bacteria or anaerobic bacteria.Oxygen deprivation did not appear to be the mode of action in thatgrowth of the strict anaerobic P. fluorescens was similar to that offacultative E. coli. Also, the growth of E. coli in the presence of thesystem of the present invention was slower than that observed in theanaerobic environment of a Gas Pak jar. Furthermore, anaerobicincubation of E. coli in the presence of the Oxysorb system inhibitedgrowth completely. The mechanism of inhibition is not yet known,however, it is likely to involve H₂ O₂ (generated by Cu²⁺ from O₂ andascorbic acid) and hypochlorous acid (HOCl) (generated by Cu²⁺ from H₂O₂ and chloride).

Ten kilograms of a mild salsa, pH 3.9, were produced and heated to 80°C., and then cooled to 24° C. To this was added 0.2% sodium ascorbate(10.1 mM) and 0.0040% (40 ppm) food grade copper gluconate (equivalentto 5.6 ppm copper =88 micromolar). This was mixed, and 176 grams wereplaced into cylindrical 7.9-cm diameter plastic tubs with 48 mlheadspace. An aluminum foil cover was heat sealed onto the tubs. Thesamples were stored under refrigeration for three months.

In the Mexican style salsa tested with the Oxysorb system, dissolvedoxygen was rapidly depleted with a concentration of 5.6 ppm copper and0.2% sodium ascorbate. However, as seen in FIG. 29, the consumption ofoxygen from the headspace is diffusion-limited. The rate constant forthe disappearance of oxygen is 0.12/day, calculated from a linearizedsemilog plot; this rate constant was used for the computer-generatedrate, shown as a solid line. The low rate of diffusion presumably arisesfrom the high viscosity of the salsa. Nevertheless, it has been foundthat the system of the present invention fully protects againstdiscoloration, off-flavors, mushiness, syneresis, and microbial growthfor more than 400 days, whereas the control reached the end of itsshelf-life after 27 days.

Whenever possible, it is advantageous to accelerate oxygen diffusion bythe following methods:

1. raising the initial storage temperature;

2. increasing the product surface;

3. agitating the product during the initial storage.

Additionally, in the case of very low diffusion rates and large headspaces, as in Mexican style salsas (176 ml salsa, 48 ml headspace in thestandard package), partial flushing with nitrogen rationally may be usedto maximize the efficiency of the system of the present invention.

The slow utilization of oxygen from the headspace of the control samplerepresents the oxidation of the product, leading to a number ofundesirable sensory attributes. The rate constant for this slow oxygenutilization was calculated to be 0.011/day, assuming first orderkinetics as in the case where the oxygen-absorbing system is used.

The above pilot plant experiment was repeated on a plant scale. It wasfound that the rate of oxygen removal from the headspace was about 30%greater than in the pilot plant, presumably because of agitation duringshipment of the product, and an increased level of ascorbate (2%ascorbic acid rather than 0.2% sodium ascorbate). The rate of oxygenremoval was slightly greater when Oxysorb was added to the cooled salsathan when it was added with the spice preblend and heat processed.However, after 420 days of refrigerated storage there was still nodifference in sensory attributes between the two variables, and bothproducts looked, smelled, and tasted significantly better than thenitrogen-flushed control. It should be noted that none of the productstreated with the oxygen absorber of the present invention were flushed.No yeast, mold, or bacterial growth occurred in the samples containingthe oxygen absorbing system of the present invention, even without anypreservatives or a modified atmosphere.

It has been observed that the sauces used on frozen and refrigeratedpizzas often oxidize during shelf-life, which causes color and flavorchanges. The sauce may become unacceptably orange and/or lose its tomatoflavor. Experiments were conducted to determine the efficacy of theOxysorb system of the present invention in stabilizing pizza-typesauces.

The formulas for the control and the Oxysorb system containing sauceswere as follows:

    ______________________________________                                                            Weight (grams)                                            ______________________________________                                        Control                                                                       Tomato paste          1559                                                    Water                 2914                                                    Soybean oil            150                                                    Spice blend            360                                                    Oxygen Scavenging System                                                      Tomato paste          1559                                                    Water                 2905                                                    Soybean oil            150                                                    Spice blend            340                                                    Ascorbic acid          20                                                     2.2% Aqueous copper gluconate solution                                                                9                                                     ______________________________________                                    

The water and oil were weighed into a beaker and mixed in a mixer.Tomato paste was added by spoonfuls, after which the preweighed spiceblend was added all at once. The sauce was mixed for 15 minutes at 1500rpm. The ascorbic acid for the test samples was added to the spiceblend. The copper gluconate was dissolved in water and added immediatelybefore the sauce was packaged.

The product was packaged in 12 oz. oxygen impermeable plastic tubs withand without shredded cheese and sealed with foil lids. The samples werestored at -12° C. and evaluated for headspace oxygen, color, and flavoronce per month.

The control product had color and flavor changes after one month and wasunacceptable after three months of storage. The products containing thesystem of the present invention had not changed after four months ofshelf-life even though headspace oxygen was removed by the Oxysorbsystem at a very slow rate, as shown in FIG. 30. The Oxysorb system alsoprovided full protection against oxidative damage in the presence ofcheese, an ingredient that normally greatly reduces shelf-life of pizzasauces by elevating the pH.

Guacamole is not shelf-stable primarily due to the instability of thecolor of the avocado when exposed to oxygen. The failure modes ofcurrently available guacamole arise from four different reactions:

1. Polyphenol oxidase catalyzes enzymatic browning within a few hours ofproduct preparation. This reaction is completely dependent on theavailability of oxygen. The system of the present invention removes alldissolved oxygen within two minutes, and was found to completely inhibitthe discoloration of guacamole for at least 80 days.

2. The high content of unsaturated lipids in avocados accelerates theonset of rancidity. By removing oxygen, the system stops the generationof off-flavors.

3. After about two months of shelf-life, oxidative damage to guacamolealso manifests itself in the form of syneresis. This phenomenon arisesfrom the oxidation of tomato particulates which causes cell wall injuryand release of water. As above, by removing oxygen, the system of thepresent invention maintains textural integrity.

4. Growth of microorganisms gives rise to large gas pockets inguacamole, ballooning of the tub, and off-flavors. The system of thepresent invention strongly inhibits bacterial, yeast and mold growth,maintains a uniform product texture, and achieves a visible vacuum inthe packaging tub due to removal of all headspace oxygen.

The guacamole was formulated as follows:

                  TABLE III                                                       ______________________________________                                        FORMULATION OF GUACAMOLE                                                                 %                                                                  INGREDIENT   CONTROL    OXYGEN SCAVENGER                                      ______________________________________                                        Red Onions   4.92       4.92                                                  Tomatoes     21.88      21.88                                                 Avocadoes    63.20      62.70                                                 Garlic Cloves                                                                              0.25       0.25                                                  Olive Oil    2.12       2.12                                                  Lemon Juice  7.63       7.63                                                  Ascorbic Acid                                                                              0          0.50                                                  Copper Gluconate                                                                           0          0.004                                                 ______________________________________                                         A.sub.w = 0.92                                                                pH = 4.1                                                                 

Each sample (160 ml) was weighed into 6-oz. oxygen-impermeable tub and afoil lid was hot sealed in place. The resulting headspace wasapproximately 50 ml. All samples were then stored in the refrigerator.

The produce was evaluated after 80 days, with the following results:

                  TABLE IV                                                        ______________________________________                                        GUACAMOLE EVALUATION AFTER 80 DAYS                                                                    OXYGEN                                                            CONTROL     SCAVENGER                                             ______________________________________                                        Color         Dark green/brown                                                                            light green                                       Odor          Sour, awful   fresh                                             Flavor        not determined                                                                              fresh                                             % Headspace O.sub.2                                                                         1.5*          1.3                                               Pressure in tub                                                                             Ballooning    Vacuum                                            Texture       Large gas pockets                                                                           Uniform                                           Visible mold growth                                                                         +             -                                                 Total plate count/g                                                                         480,000       6,000                                             Overall acceptability                                                                       No            Yes                                               ______________________________________                                         *The low headspace oxygen of the control indicates that extensive             oxidative damage to the prouct has occurred.                             

Another experiment was designed to demonstrate visually that the systemof the present invention does inhibit polyphenol oxidase activity inpotatoes. When potatoes are pureed and exposed to oxygen, they rapidlyturn a reddish-brown within a few minutes and finally a black colorafter 7 days. When subjected to a chemical agent such as sodiumbisulfite, this browning does not occur. This color change can also beprevented by the system of the present invention. A control of water andpotato was pureed in a blender and placed into a screw top jar, filledto overflowing to minimize headspace. The same was done to a sample ofpotato and 40 ppm copper gluconate and 0.1% ascorbic acid in water. Thejars were refrigerated and inverted frequently to disperse any foamformed.

The control started browning immediately upon processing. Browning dueto the release of the polyphenol oxidase continued to increase until thesolution became nearly black over a period of seven days. The potatoesexposed to the system of the present invention remained an off-whitecolor, but the surface foam darkened slightly. When the samples wereinverted to distribute the foam, the browning disappeared. Thisdifference in the browning was also quantitated spectrophotometricallyby measuring the absorption at 475 nm, as shown in FIG. 31.

Another experiment was conducted to determine if the system of thepresent invention could delay or prevent textural degradation ofpotatoes stored in water. Raw potatoes were peeled, cubed, and immersedin water and in solutions of 40 ppm copper gluconate and 0.1% or 0.5%ascorbic acid. The samples were kept in uncovered glass mason jars atroom temperature.

After one day, the control was a little cloudy, indicating microbes weregrowing. By day 2 the control had foam on top of the water surface, andthe water was cloudy and turbid. Meanwhile, the other samples were clearand without foam. By day 3, the control smelled putrid and the potatoeswere floating on top. The texture of the potatoes was gooey and sticky,and they had lost the cubed shape as they melted together. Theyexhibited a moldy color, and the water was yellowish and turbid. The0.1% ascorbate sample was beginning to show slight turbidity and therewere some bubbles on top, although there was no odor. The 0.5% solutionhad clear water, but some bubbles on top. On day 4, the 0.1% solutionhad some cubes floating, and murky water, while the 0.5% solution wasstill clear. After 7 days, the 0.5% solution had some turbidity, andsome bubbles on top. After two weeks, the water was dark yellow, butnone of the potatoes were floating, and they retained their shape andtexture. After 16 days, some potatoes were floating and getting gooey.Sodium bisulfite, an inhibitor of enzymatic browning, had no effect atall on these chemical and microbiological events.

                  TABLE V                                                         ______________________________________                                        MICROBIOLOGICAL EVALUATION OF POTATO WATER                                                 Total Plate                                                                   Count*  Coliforms*                                                                              Anaerobes*                                     ______________________________________                                        control        2.8 × 10.sup.8                                                                    >2400     3.1 × 10.sup.7                       0.1% AA + 40 ppm Cu.sup.2+                                                                   1.7 × 10.sup.8                                                                    >2400     3.2 × 10.sup.7                       0.5% AA + 40 ppm Cu.sup.2+                                                                   2.5 × 10.sup.7                                                                      93      1.5 × 10.sup.7                       ______________________________________                                         *(microorganisms/ml)                                                     

Aseptically packaged corn in a microwaveable plastic container exhibitsexcellent microbiological stability when stored at 22° C., but it turnsgrey over 90 days. This greying phenomenon has been demonstrated to be adirect result of oxidative damage, presumably by oxygen dissolved in thewater and present in the headspace.

The system of the present invention has been found to remove all of thedissolved oxygen within three minutes and the headspace oxygen withintwo to three days, because of the slow diffusion of oxygen into thewater. However, during these initial two to three days, the actualconcentration of oxygen in the water will be close to zero. Furthermore,the system will remove any oxygen permeating through the packagingmaterial during storage. Therefore, the addition of the system shouldprotect the corn from greying and extend its ambient shelf-life to atleast two years. This technology is also applicable to the preservationof canned peas, asparagus, and other vegetables.

One problem associated with ready-to-spread frosting that limits itsshelf-life is mold. To prevent mold growth, current products now useBHA, sorbate, and citric acid. The citric acid keeps the pH low andmakes the sorbate effective. Other problems associated with thesefrostings are off-flavor development and color changes.

The system of the present invention was tested in ready-to-spreadfrostings packed in substantially oxygen-impermeable plastic tubs. Afterthree weeks, these were pulled, and a microbial analysis was conducted.The results in Table VI indicate that the system of the presentinvention inhibits yeast growth when added directly to tho food product,and decreased rancidity of the product, as indicated by peroxide values(PV) and malondialdehyde (MDA) results:

                  TABLE VI                                                        ______________________________________                                        EVALUATION OF FROSTING                                                        Sample      Yeast/gram                                                                              MDA (mg/kg) PV (mg/kg)                                  ______________________________________                                        control     660000    0.21        3.1                                         Oxysorb pouch                                                                              95000                                                            Oxysorb dissolved                                                                          <100     0.15        0.4                                         directly in RTS                                                               ______________________________________                                    

The Oxysorb system is added in an amount effective to preventobjectionable and deleterious amounts of oxygen degradation and/ormicrobial growth. The measurements of both end results are known in theart e.g., organoleptically and plate counts, respectively.

The amounts of each system component and the system will depend onseveral factors. These factors include such things as: headspace amount,oxygen in the headspace, product to be protected, dissolved andcontained oxygen, packaging permeability, shelf-life, storagetemperature, etc. Government regulations and taste of the system canalso limit the amounts. Thus, the amounts needed can vary widely but canbe easily determined.

For food products, copper in the range of between about 1 ppm and about7 ppm and ascorbate in amounts in the range of between about 0.05% andabout 1% have been found effective for use in relatively high oxygenimpermeable packaging.

The foregoing description of the specific embodiment will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and therefore such adaptations and modifications are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description and not oflimitation.

What is claimed is:
 1. In a packaged ingestible product comprising asubstantially non-oxygen permeable package and, contained therein, aningestible product and an oxygen scavenging composition, the improvementwherein said oxygen scavenging composition comprises a solution of areducing agent and a dissolved species of copper, said dissolved copperbeing present in an amount so as to provide, with respect to the totalproduct within the package, from about 1 to 7 parts per million ofcopper as copper ion.
 2. A packaged product in accordance with claim 1,wherein said reducing agent is ascorbic acid or salts or esters thereof,sodium sulfite, cysteine or catechol.
 3. A packaged product inaccordance with claim 1, wherein said oxygen scavenging compositionfurther includes a chelating agent.
 4. A packaged product in accordancewith claim 3, wherein said chelating agent is selected from the groupconsisting of glycine, citric acid, EDTA, phosphate compounds andmixtures thereof.
 5. A packaged product in accordance with claim 1,wherein said ingestible product is a food or a pharmaceutical.
 6. Apackaged product in accordance with claim 1, wherein said oxygenscavenging composition is dissolved in a vehicle selected from the groupconsisting of water, propylene glycol, glycerol, ethanol, fat andmixtures thereof.
 7. A packaged product in accordance with claim 1,wherein said package is a jar or bottle with a lid or cap and the oxygenscavenging composition is incorporated in the lid or cap.
 8. A packagedproduct in accordance with claim 1, wherein said oxygen scavengingcomposition in enrobed in an oxygen- and water-permeable material.
 9. Apackaged product in accordance with claim 1, wherein said copper ispresent in an amount of about 3 parts per million.
 10. A packagedproduct in accordance with claim 6, wherein said vehicle is ethanol. 11.A packaged product in accordance with claim 1, wherein said oxygenscavenging composition comprises ascorbyl-6-palmitate and coppercaprylate.
 12. A packaged product in accordance with claim 2, whereinsaid reducing agent is ascorbic acid or a salt or ester thereof.
 13. Apackaged product in accordance with claim 6, wherein said vehicle isfat.
 14. A packaged product in accordance with claim 6, wherein saidvehicle is water.
 15. A packaged product in accordance with claim 1,wherein said product is one containing fat, water or both fat and waterand said reducing agent and said copper are dissolved in said fat and/orwater of said product.
 16. A packaged product in accordance with claim1, wherein said product is a food.
 17. A packaged product in accordancewith claim 15, wherein said product is a food.
 18. A packaged product inaccordance with claim 1, wherein said copper is in the form of a Cu⁺ orCu²⁺ ion.